The long term objective of this project is to understand the individual molecular interactions responsible for tight binding, specificity and efficient catalysis in the Aspartic Proteinase class of enzymes. These proteolytic enzymes are found in a wide diversity of organisms and play important roles in many physiological processes: human enzymes pepsin and gastricsin in digestion; renin in regulation of blood pressure; and cathepsin D in intracellular protein turnover and in proenzyme and prohormone processing. The retroviral aspartic proteinases are indispensable for the virus life cycle as they process precursor polyproteins into individual functional entities. Crystallographic analysis has shown that these enzymes share a common three-dimensional structure with an extended binding cleft capable of interacting with seven to eight amino acid residues and situated at the interface between the N-terminal and C-terminal domains in the eukaryotic enzymes and at the dimer interface in the retroviral systems. This cleft interacts with ligands in a common fashion. A remarkably similar pattern of hydrogen bonding to the backbone of bound peptides has been observed. However, specific interactions between individual amino acid side chains in the various enzyme subsites are different. Each enzyme, therefore, has subtly different specificity based on binding at individual "secondary: subsites. This program presents a combined approach in which synthesis of new oligopeptide substrate or inhibitor sequences will be joined with protein engineering, expression and purification of recombinant enzymes to explore the nature of the multiple interactions occurring in the cleft. Enzyme residues identified from crystal structures as being located in a specific subsite will be altered to test specific hypotheses on binding interactions. The mutant proteins will be thoroughly characterized. The kinetic properties of the enzymes will be studied using a chromogenic assay with a series of peptide substrates with sequence changes in the position of the substrate complementary to the change in the enzyme subsite. By examination of a group of inhibitors with regular changes in sequence at the specific position under examination additional information will be obtained to test specific structure-function hypotheses. The information gained in this study will add to the understanding of enzyme-substrate interactions in general and will contribute to the design of selective, therapeutically useful enzyme inhibitors for the Asparatic Proteinase family.